Abstract

Respiratory infection with Francisella tularensis (Ft) is characterized by a muted, acute host response, followed by sepsis-like syndrome that results in death. Infection with Ft establishes a principally anti-inflammatory environment that subverts host-cell death programs to facilitate pathogen replication. Although the role of cytokines has been explored extensively, the role of eicosanoids in tularemia pathogenesis is not fully understood. Given that lipoxin A4 (LXA4) has anti-inflammatory properties, we investigated whether this lipid mediator affects host responses manifested early during infection. The addition of exogenous LXA4 inhibits PGE2 release by Ft-infected murine monocytes in vitro and diminishes apoptotic cell death. Tularemia pathogenesis was characterized in 5‑lipoxygenase-deficient (Alox5-/-) mice that are incapable of generating LXA4 Increased release of proinflammatory cytokines and chemokines, as well as increased apoptosis, was observed in Alox5-/- mice as compared with their wild-type counterparts. Alox5-/- mice also exhibited elevated recruitment of neutrophils during the early phase of infection and increased resistance to lethal challenge. Conversely, administration of exogenous LXA4 to Alox5-/- mice made them more susceptible to infection thus mimicking wild-type animals. Taken together, our results suggest that 5-LO activity is a critical regulator of immunopathology observed during the acute phase of respiratory tularemia, regulating bacterial burden and neutrophil recruitment and production of proinflammatory modulators and increasing morbidity and mortality. These studies identify a detrimental role for the 5-LO-derived lipid mediator LXA4 in Ft-induced immunopathology. Targeting this pathway may have therapeutic benefit as an adjunct to treatment with antibiotics and conventional antimicrobial peptides, which often have limited efficacy against intracellular bacteria.

Lipid mediators PGE2 and LXA4 are reciprocally regulated during the course of Ft LVS infection.

(A) Wild-type C57BL/6 mice were infected with 1000 CFU of Ft LVS, and the BALF cells were isolated from individual mice on d 2 postinfection. The BALF cells were stained with Nile Red (0.1 ng/ml) and cytospun. The cells were fixed in 3.7% formaldehyde and were analyzed by confocal microscopy. The results are representative of 6 individual mice. Levels of PGE2 (B) and LXA4 (C) during the course of Ft infection were measured in lung homogenates by mass spectrometry. Data are presented as the means ± sem from 3 independent experiments (n = 6 mice/group; 18 mice total). *P < 0.05, ***P < 0.001. All results shown were subjected to 1-way ANOVA with Bonferroni’s posttest.

(A) Wild-type C57BL/6 BMDMs (2.5 × 105 cells/well) were seeded into 24-well plates and infected with Ft LVS at an MOI of 100. Exogenous LXA4 (1 µM) was added at the time of infection to the cells. Supernatants were collected after incubation for 24 h at 37°C and were analyzed for the presence of PGE2. **P < 0.01, ***P < 0.001. (B) Wild-type C57BL/6 BMDMs (1 × 106 cells/well) were infected with Ft LVS at an MOI of 100. Exogenous LXA4 (1 µM) was added to cells at the time of infection, and after incubation for 24 h at 37°C, the cells were stained with TUNEL to quantify the percentage of cells undergoing apoptosis. ***P < 0.001. Data are presented as the means ± sem from 3 independent experiments. All results shown were subjected to 1-way ANOVA with Bonferroni’s posttest.

(A) Wild-type (Alox5+/+) and Alox5−/− C57BL/6 BMDMs (2.5 × 105 cells/well) were seeded into 24-well plates and infected with Ft LVS at an MOI of 100. Cells were incubated at 37°C, and supernatants were collected at various times and analyzed for the presence of PGE2. ***P < 0.001. (B) Wild-type (Alox5+/+) and Alox5−/− C57BL/6 BMDMs (1 × 106 cells/well) were seeded into 6-well plates and infected with Ft LVS at an MOI of 100 and were incubated for 24 h at 37°C. The cells were stained with TUNEL to quantify the percentage of undergoing apoptosis. ***P < 0.001. Data are presented as the means ± sem from 3 independent experiments. All results shown were subjected to One-way ANOVA with Bonferroni’s Posttest. Asterisks immediately above columns indicate a significant difference compared with their corresponding control while asterisks above a bracket indicate a significant difference between 2 experimental groups.

The inhibitor of the 5-LO pathway, MK-886, results in lower bacterial burden.

(A) Wild-type C57BL/6 mice were infected i.n. with Ft LVS alone or with doses of MK-886 (5 mg/kg) at the time of infection and, thereafter, every 24 h. The infected mice were sacrificed at different times as shown and were analyzed for the presence of LXA4 by ELISA. (B) The lung homogenates were prepared and plated for determination of bacterial burden. *P < 0.05, **P < 0.01. Results shown are the means ± sem and are representative of 2 independent experiments (n = 6 mice/time point; 12 mice total). Asterisks immediately above columns indicate a significant difference compared with its corresponding control, whereas asterisks above a bracket indicate a significant difference between 2 experimental groups.